Mechanism of Binding to Ebola Virus Glycoprotein by the ZMapp, ZMAb, and MB-003 Cocktail Antibodies

Abstract

Cocktails of monoclonal antibodies (MAbs) that target the surface glycoprotein (GP) of Ebola virus (EBOV) are effective in nonhuman primate models and have been used under emergency compassionate-treatment protocols in human patients. However, the amino acids that form the detailed binding epitopes for the MAbs in the ZMapp, ZMAb, and the related MB-003 cocktails have yet to be identified. Other binding properties that define how each MAb functionally interacts with GP—such as affinity, epitope conservation, and epitope accessibility—also remain largely unknown. To help define how each MAb interacts with GP, here we used comprehensive alanine-scanning mutagenesis (shotgun mutagenesis), neutralization escape, and whole virion binding to define each MAb's specific epitope, epitope accessibility, epitope conservation, and apparent affinity. Each of the six therapeutic MAbs binds nonidentical epitopes in the GP base, glycan cap, or mucin-like domain. Their apparent affinity, epitope complementarity, and epitope accessibility helps explain why MAbs 4G7 and 13C6 are more protective than 2G4 and 1H3. The mucin-like domain MAbs 6D8 and 13F6 bind with the strongest apparent affinity, helping to explain their effectiveness in vivo despite their inability to neutralize virus.

Importance: Ebola virus disease (EVD) can be caused by four different filovirus family members, including Ebola virus (EBOV), which infected 10 times more people in western Africa over the last year than all previous EVD outbreaks combined, with a number of cases distributed across the globe by travelers. Cocktails of inhibitory monoclonal antibodies (MAbs), such as ZMAb, MB-003, and in particular ZMapp, have demonstrated in animal models some of the most significant therapeutic potential for treating EVD, and in 2014, 15 patients were treated with ZMapp or ZMAb under compassionate-use protocols. Here, we have defined the epitope features for the most important therapeutic MAbs against EBOV developed to date. Defining the epitopes and binding characteristics for these MAbs, as well as the commonly used reference MAb KZ52, helps explain their breadth of reactivity against different ebolavirus species, predict viral evasion against these MAbs, and design new cocktails of MAbs with improved complementarity.

FIG 1

Shotgun mutagenesis epitope mapping of EBOV MAbs. (A) A mutation library for EBOV envelope protein encompassing 641 GP mutations was constructed in which each amino acid was individually mutated. Residues were changed to alanine (with alanine residues changed to serine) to provide a controlled method for defining the side chain contributions of each residue. Each well of each mutation array plate contains cells expressing one mutant with a defined substitution. A representative 384-well plate of reactivity results is shown. Eight positive (wild-type GP) and four negative (mock-transfected) control wells are included on each plate. Wells low in signal (<20% of wild type) are colored red. (B) Human HEK-293T cells expressing the EBOV envelope mutation library were tested for immunoreactivity with MAb KZ52 and a control MAb (13C6 shown) and measured using an Intellicyt high-throughput flow cytometer. The highlighted region identifies clones with high GP expression but low KZ52 binding, and clones that were confirmed as critical are shown in red. Other clones in the highlighted region were not confirmed as critical based on likely misfolding. (C) Mutation of five individual residues reduced KZ52 binding (red bars) but did not affect binding of most other MAbs (gray, black, and white bars). Error bars represent the mean and range (half of the maximum minus minimum values) of at least two replicate data points.

FIG 2

Epitope mapping of EBOV cocktail MAbs. GP residues critical for binding each MAb are shown in green on one GP monomer (left diagram of each panel) and the GP trimer (right diagram of each panel) of the EBOV Δmucin GP structure (PDB accession no. 3CSY) (24) for MAbs 2G4 (A), 4G7 (B), 13C6 (C), 1H3 (D), 6D8 (E), and 13F6 (F). The locations of residue P279 (D) and residues in the mucin-like domain (E and F) are approximated, as the structures of these regions (residues 279 to 298 and mucin-like domain residues 313 to 464) are not yet solved. Also indicated (top right of each panel) are the cocktails that contain the MAb.

FIG 3

Mapping of MAb KZ52 identifies the energetically critical epitope residues of the interaction. (A) Residues are shown on the crystal structure of EBOV Δmucin GP (PDB accession no. 3CSY) (24). One monomer is shown, with GP1 colored gold and GP2 colored red. KZ52 contact residues identified from crystallographic analysis (left diagram) are shown in blue. The residues identified here by mutagenesis are shown in green (right diagram). (B) The KZ52 epitope (residues identified here in green) forms a conformational epitope that lies at the interface of GP1 and GP2 at the base of the GP trimer (monomers indicated as I, II, III).

FIG 4

Visualization of epitopes within the EM footprint. Visualization of epitopes obtained by mutagenesis with the Fab footprints from electron microscopy (25) suggest that the critical residues identified by mutagenesis are the energetically critical hot-spot residues at the center of the MAb epitope. The EM footprint residues (purple) and mutagenesis hot-spot residues (green) are shown in the context of the Fab EM density information (solid structures). Docking of each MAb to GP was obtained by fitting the EBOV GP crystal structure (25) into the structural information for the EM reconstructions of Fabs bound to EBOV GP, identified by the EMDataBank ID emd-6151 (2G4) (A), emd-6152 (4G7) (B), emd-6152 (13C6) (C), or emd-6150 (1H3) (D) (25). Insets show the view of the footprints and hot spots from the angle of the Fab. The location of residue P279 (D) is approximated, as the structure of this region (residues 279 to 298) is not yet solved.

FIG 5

Neutralization escape studies validate epitope residues. MAbs were tested for their ability to neutralize the infectivity of lentiviral reporter pseudotypes with full-length EBOV GP. Reporter pseudotypes were preincubated with MAbs, and infection of HEK-293T target cells was detected by the expression of Renilla luciferase. (A) MAbs 13C6, 1H3, 6D8, and 13F6 demonstrated no neutralization at the concentrations tested. (B to D) Known neutralizing MAbs KZ52 (B), 2G4 (C), and 4G7 (D) were tested for neutralization of reporter pseudotypes with wild-type (WT) EBOV GP and with GPs containing mutants critical for binding by KZ52 (D552A, G553A), 2G4 (G553A), and 4G7 (D552A). Data points represent the mean of three replicates (± standard deviation), and data are representative of two independent experiments.

FIG 6

MAb cross-reactivity with Ebolavirus species GP. MAbs were tested by flow cytometry for immunoreactivity with HEK-293T cells transfected with constructs expressing GP from EBOV, BDBV, TAFV, or SUDV, an EBOV Δmucin GP construct, or empty vector. All ebolavirus GPs were fully reactive with a control MAb against the V5 epitope tag included on the C terminus of each protein as well as with other anti-GP MAbs (not shown). Data shown represent the mean and standard deviation of at least four data points. RFU, relative fluorescence units.

FIG 7

Conservation of epitope residues. (A) Diagram of EBOV GP1 and GP2 showing the location of 19 residue changes (red circles) that have occurred between the EBOV Makona isolate from the 2013-to-present day outbreak and the 1976 EBOV Yambuku-Mayinga isolate (45). The locations of critical epitope residues identified in our study are shown in green. SP, signal peptide; TM, transmembrane region. (B) Alignment of the GP domains representing the five species of ebolaviruses (EBOV, BDBV, TAFV, SUDV, and RESTV), showing the critical epitope residues for ZMapp, ZMAb, and MB-003 MAbs. The epitope residues identified for the indicated MAbs are highlighted green, and the locations of residues altered by changes in EBOV are marked with red circles. Conservation among the five ebolaviruses is shown below the alignment: asterisks indicate complete conservation, colons indicate conservative mutations, and periods indicate semiconservative mutations.

FIG 8

Relative binding affinities of EBOV cocktail MAbs. ELISAs using VLPs pseudotyped with EBOV wild-type GP (A) or EBOV Δmucin GP (B) were used to test 2-fold dilutions of each MAb to compare apparent binding affinities against GP on virions. VLPs were also tested for reactivity with a V5 antibody against a V5 epitope tag incorporated onto the C terminus of EBOV WT GP and EBOV Δmucin GP (dotted line).

FIG 9

Inhibitory epitopes on EBOV GP. Residues identified as critical for the MAbs assayed in this study are represented on the crystal structure of EBOV Δmucin GP (PDB accession no. 3CSY) (24). (A) All epitopes. (B) MB-003 cocktail epitopes. (C) ZMAb cocktail epitopes. (D) ZMapp cocktail epitopes. For reference, the epitope for KZ52 is highlighted in panel A, overlapping the 2G4 (red outline) and 4G7 (green) epitopes. For each cocktail (B to D), a value for the percent survival is indicated, obtained from a direct comparison of the results of the cocktails in guinea pigs (5-mg doses given 3 days postinfection) (18).